College of Forestry, Northwest A&F University, Yangling, Shaanxi, 712100, China.
Mol Biol Rep. 2019 Jun;46(3):3083-3092. doi: 10.1007/s11033-019-04746-2. Epub 2019 Mar 11.
Two novel miRNAs were selected from a pre-constructed RNA library of Populus szechuanica infected with the foliar rust fungus Melampsora larici-populina in order to detect the genes regulated as targets of the miRNAs novel_mir_11 and novel_mir_357. The novel miRNAs were identified from P. szechuanica using stem-loop methods and their precursors were able to fold into a complete stem loop structure. The predicted target genes of the novel miRNAs were verified with RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5'RACE). The full-length sequences of target genes, RPM1 and RPS2/5, in P. szechuanica were obtained through rapid amplification of cDNA ends (RACE) and officially named PsRPM1 and PsRPS2/5. These genes contain nucleotide binding site-leucine-rich repeats (NBS-LRR) domains typical of resistance genes. The expression levels of miRNAs and their target genes in different periods post infection were analysed with quantitative real-time PCR (qRT-PCR). After infection with the foliar rust fungus, the expression levels of the novel miRNAs and their target genes were dynamic. Both novel_mir_11 and novel_mir_357 negatively regulated the expression of their target genes. In this study, the regulatory effects of two novel miRNAs through their target genes were characterized to provide further mechanistic information regarding the interaction between Populus and a foliar rust fungus. Results of this study improve our understanding of the defence response mechanisms of Populus and will stimulate future work to characterize strategies to prevent and control Populus diseases.
为了检测作为 miRNA novel_mir_11 和 novel_mir_357 靶基因调控的基因，我们从感染杨树叶锈菌的四川杨构建的 RNA 文库中选择了两个新的 miRNA。使用茎环方法从 P. szechuanica 中鉴定出新的 miRNA，其前体能够折叠成完整的茎环结构。miRNA 的预测靶基因通过 RNA 连接酶介导的 5' 快速扩增 cDNA 末端 (RLM-5'RACE) 进行验证。通过快速扩增 cDNA 末端 (RACE)，获得了 P. szechuanica 中靶基因 RPM1 和 RPS2/5 的全长序列，并正式命名为 PsRPM1 和 PsRPS2/5。这些基因含有核苷酸结合位点-富含亮氨酸重复 (NBS-LRR) 区域，是抗性基因的典型特征。用定量实时 PCR (qRT-PCR) 分析不同感染后时期 miRNA 和其靶基因的表达水平。感染杨树叶锈菌后，新的 miRNA 和其靶基因的表达水平呈动态变化。novel_mir_11 和 novel_mir_357 均负调控其靶基因的表达。在本研究中，通过靶基因对两个新 miRNA 的调控作用进行了表征，为杨树与叶锈菌相互作用的机制提供了进一步的信息。本研究结果提高了我们对杨树防御反应机制的理解，并将激发未来的工作，以表征预防和控制杨树病害的策略。
